Rainbow trout liver microsomes were used to study the O-demethylation and r
ing hydroxylation of 4-methoxyphenol (4-MP) (4-hydroxyanisole) at 11 and 25
degrees C by directly measuring the production of the primary metabolite h
ydroquinone (HQ), 4-methoxycatechol (I-MCAT), and additional metabolites. A
n HPLC method with integrated ultraviolet (UV) and electrochemical detectio
n (ECD) was developed for metabolite identification and quantification at l
ow concentrations. Sample handling with appropriate buffers, solvents, low
temperature and light prevented loss of extremely labile metabolites. Satur
ation kinetics for the production of HQ via O-demethylation of 4-MP (0.66-4
0 mM) was never achieved, with substrate solubility being the limiting fact
or. The linear rate of HQ formation at 11 degrees C was 22.0 +/- 2.2 (coeff
icient +/- S.E., r(2) = 0.91) pmol min (-1) per mg protein per mM substrate
, and at 25 degrees C was 34.0 +/- 1.3 (r(2) = 0.99) pmol min (-1) per mg p
rotein per mM substrate. The second major microsomal metabolite 4-MCAT was
also identified, with linear rates of ring hydroxylation determined to be 1
9.0 +/- 1.6 (r(2) = 0.94) and 17.2 +/- 0.5 (r(2) = 0.99) pmol min (-1) per
mg protein per mM substrate at 11 and 25 degrees C, respectively. Unlike HQ
production,. the rate of 4-MCAT production was found to be similar at the
two temperatures when linear formation rates were corrected for the effect
of temperature on substrate and product solubility at 11 degrees C. Measure
ment of 'freely dissolved ed fraction was essential to the accurate determi
nation of ring hydroxylation and O-demethylation reaction rates in rainbow
trout microsomes incubated at physiological temperature. Experimental condi
tions were shown to affect dissolved 4-MP and HQ at 11 degrees C (verified
using microdialysis) while not altering substrate and product levels at 25
degrees C. Small but detectable levels of 1,4-benzoquinone were observed in
4-MP microsomal incubations. 1,2, 4 -Trihydroxybenzene was also detected,
with possible routes of production through hydroxylation of HQ or O-demethy
lation of 4-MCAT. A metabolic scheme for bioactivation of 4-MP is proposed
and the significance of observed metabolic conversions in rainbow trout mic
rosomes discussed in relation to aquatic toxicity of 4-MP. (C) 1999 Elsevie
r Science B.V. All rights reserved.